The goal of this proposal is to identify and characterize endogenous substrates of the insulin receptor tyrosine protein kinase. The beta subunit of the insulin receptor is a tyrosine kinase. The connection between the tyrosine kinase activity of the occupied insulin receptor and the physiological actions of insulin is not yet known. This project aims at elucidating this relationship using cells that express a high content of insulin receptors and are sensitive to physiological concentrations of insulin. The initial aim is the identification of phosphotyrosine containing proteins which are increased in cells stimulated by insulin. Phosphotyrosine containing proteins which are increased in cells stimulated by insulin. Phosphotyrosine containing proteins are rare, constituting less than 0.2% of the phosphoproteins in cells. This proposal takes advantage of several recently developed procedures for identifying cellular substrates. (1) Antibodies to phosphotyrosine. These antisera permit detection of phosphotyrosine containing proteins either by immunoblotting or by immunoprecipitation of labelled proteins, followed by resolution on SDS-polyacrylamide gel electrophoresis. (2) Antibodies prepared to peptides corresponding to the deduced amino acid sequence of the human insulin receptor. Since the peptides used to elicit the antibodies include tyrosine residues that are phosphorylated by the receptor kinase, it is possible that endogenous substrates will have similar amino acid sequences and would be recognized by these antibodies. (3) Polyclonal and monoclonal antibodies prepared to the intact receptor can be used to immunoprecipitate substrates associated with the receptor protein. The projected study also makes use of transfected CHO cell lines, developed in this laboratory, which stably express normal and mutant human insulin receptors. Once substrates are identified, they will be eluted from gel bands and used either for raising specific monoclonal antibodies or polyclonal antibodies or for amino acid sequencing. Antibodies to these putative substrates of the insulin receptor will be used to further purify, characterize and, possibly, clone the genes for selected proteins. Once substrates have been purified, this project will examine the function of these substrates. Our long term goal is in vitro reconstitution of one or more of the early actions of insulin utilizing the biochemical pathways currently under investigation in the laboratory (e.g. S6 kinase and hexose transport activation). The project thus addresses the possible role of tyrosine kinase activity in the physiological consequences of insulin action.